Technical Abstract:
We have identified the complementary and genomic DNA sequences of the novel growth hormone-releasing peptide, ghrelin, in the cichlid fish (tilapia Oreochromis niloticus). The tilapia ghrelin precursor cDNA was 855 bp long, consisting of 119 bp of 5'-untranslated region, 324 bp open reading frame, and 412 bp of 3'-untranslated region. The serine residues at positions 2 and 3 of the "active core" (GSSF) of the ghrelin mature peptide are conserved between tilapia and the mammalian species. The tilapia ghrelin gene has four exons and three introns and resembles the structure of other known ghrelin genes. RT-PCR analysis revealed ghrelin mRNA predominantly expressed in the stomach but absent in the brain, pituitary, heart, kidney, ovary, and testis. Real-time PCR analysis showed an age/body size dependent increase in gastric ghrelin, which stagnated at the onset of sexual maturation. Ghrelin mRNA levels were unchanged in food-deprived sexually mature animals but were significantly higher in females compared to males. Injections of ghrelin into the brain significantly increased pituitary growth hormone (GH) mRNA levels and plasma GH levels. Immunocytochemical analysis showed ghrelin had no effect on gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neuronal cell numbers, cell optical density, but significantly decreased growth hormone-releasing hormone (GHRH) cell numbers. The present study shows that the structure of ghrelin peptide is highly conserved, and the differences in somatic and gonadal growth in tilapia could be a consequence of age- and sex-related synthesis of gastric ghrelin. Further, we show that the action of ghrelin on GH synthesis and release could be at the level of the pituitary or the hypothalamus via the regulation of GHRH neurons.